Project Summary (<=30 lines) Cryogenic electron microscopy (cryoEM) has become a break-through structural biology method for scientists to study biological specimens in their native environment. An increasing number of scientists are eager to use this powerful technology since it provides a new way to study significant outstanding biology problems that were impossible to explore just a few years ago. However, cryoEM is complex and involves, in addition to theoretical background, operating multiple devices that require a strict protocol and sequence as well as accurate and trained hand-eye coordination as mistakes in operation would risk instrument downtime and costly repairs. Becoming a proficient cryoEM user requires extensive hands-on practice under the supervision of expert users and current best-practice approaches to train users involve thorough and repeated hands-on observation, supervision and practice on site. However, due to the research demands for machine and staff time, as well as potentially high cost, novice users often have limited access to such facilities and are therefore restricted in their training and in opportunities for productive application of cryoEM in their research. The goal of this project is to develop a virtual reality (VR) augmented interactive training system, CryoVR, to provide virtual hands-on, self- paced cryoEM training to familiarize novice users with operational procedures, thereby reducing the need for practice with costly cryoEM devices; using this approach to complement existing theoretical and protocol training will save time, reduce cost, and lower the risk of impairing cryoEM instruments. This VR approach will be available online, enabling training far before practical training necessitates time on the instrument. In addition, return users, who may have last used cryoEM months or years previously, can use CryoVR to rehearse and refresh their skills before returning to the facility. We pursue this goal with three aims: Aim 1) Use state-of-the- art VR devices to address the needs for orienting new users to cryoEM instruments (e.g. plunge freezers, electron microscopes, etc.), developing mastery over sequence of operations and method of using instruments to perform specific key tasks (e.g. glow discharging grids, plunge-freezing sample grids, cryo-transfer of grids to TEM column, TEM operation, etc.), and providing sufficient review for returning non-expert users; Aim 2) In order to ensure the accuracy and usability of CryoVR, we will evaluate our VR instructional materials, their impact and outcomes through the collection of voluntary participant feedbacks; and Aim 3) In order to maximize availability of CryoVR, we will establish distribution methods for both online and downloadable software. The proposed approach is innovative in cryoEM training and will be effective and efficient due to the stability and applicability of modern VR technologies, practices, and theories. The project is significant as it will greatly improve the efficiency, and reduce the cost and risk of cryoEM training, which in turn leads to increased capacity for training new users. The low-cost VR hardware and freely accessible software will benefit all cryoEM facilities and their users on a national and international scale.